Ultra rapid freezing for cell cryopreservation

ABSTRACT

A method for preserving biological material includes the steps of placing the biological material in thermal contact with a cryogenically coolable environment, applying radiant energy to the biological material to maintain the temperature of the biological material at physiological temperatures, cooling the surrounding environment to a temperature below the glass phase transition temperature of the biological material, and rapidly stopping the application of radiant energy to the biological material. The method produces cooling rates so rapid that the biological material is vitrified without an opportunity for ice crystals to form.

CROSS-REFERENCE TO RELATED APPLICATIONS

Not Applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable.

FIELD OF THE INVENTION

The present invention relates to a method and apparatus forcryopreservation of biological material using ultra rapid freezing.

BACKGROUND OF THE INVENTION

With recent advances in cell transplantation, tissue engineering andgenetic technologies, the living cell is becoming an importanttherapeutic tool in clinical medical care. From the use of livingartificial skin and bone material to treat burn and trauma victims, tobioartificial devices and direct transplantation of cellular material totreat the increasingly long list of genetically-based diseases, livingcells are increasingly incorporated into comprehensive treatment. Insuch a construct, the exogenous cells perform the multitude of complextasks which the diseased tissue cannot. Successful long-termpreservation and storage of mammalian cells is critical to the successof this type of medical care.

Conventional cryopreservation protocols rely on the addition ofcryoprotectants to control the formation of damaging crystalline ice inthe intracellular and extracellular liquid. The formation of ice in theextracellular liquid leads to dehydration of cells and has also beenshown to catalyze the formation of intracellular ice [Toner et al.,“Thermodynamics and kinetics of intracellular ice formation duringfreezing of biological cells,” 67 J.Applied Phys. 1582-1593 (1990)]. Theformation of intracellular ice directly damages the cell and usuallyleads to cell death.

Equilibrium and non-equilibrium cryopreservation protocols try tobalance the deleterious effects of cell dehydration, exposure of thecells to toxic cryoprotectants and the lethal formation of intracellularice so as to yield the highest possible percentage of viable cells[Mazur, “Equilibrium, Quasi-equilibrium, and non-equilibrium freezing ofmammalian embryos,” 17 Cell Biophysics 53-92 (1985)]. Protocols of thistype have been very successful for certain cell types. For example,survival rates of greater than 90% have been reported for erythrocytes[Nei, “Freezing injury to erythrocytes I. Freezing patterns andpost-thaw hemolysis,” 13 Cryobiology 278-286 (1976)], pancreatic islets[Jutte et al., “Vitrification of Human Islets of Langerhans,” 24Cryobiology 403-411 (1987)] and mouse oocytes [Karlsson et al,“Fertilization and development of mouse oocytes cryopreserved using atheoretically optimized protocol,” 11 Human Reproduction 1296-1305(1996)].

There are many cell types, however, for which acceptablecryopreservation protocols have not been developed. This is largely dueto the fact that the concentration of cryoprotectant required to avoidintracellular ice formation is too high for most cells to tolerate [Fahyet al, “Vitrification as an approach to cryopreservation,” 21Cryobiology 407-426 (1984)]. Among the important cell types for whichsuccessful and reliable freezing protocols have not been developed arehepatocytes, human oocytes, platelets and granulocytes. The fact thathuman oocytes have not been preserved successfully in spite of thesuccessful freezing of mouse oocytes illustrates another difficulty withthe current methods of cryopreservation: they are extremely dependent oncell type. Even closely related cell types behave and survivedifferently when cryopreserved.

An alternative to conventional approaches to cryopreservation byfreezing with high levels of cryoprotectant is vitrification, i.e.,solidification of a liquid into an amorphous or glassy state as opposedto the crystalline state. Unlike the liquid-to-crystal transition, theliquid-to-glass transition is generally believed not to have any adversebiological effects. This is because there is no elevation in electrolyteconcentration, no ice crystals to cause mechanical damage, and nopotentially damaging osmotic shifts during the vitrification of cellsuspensions.

It appears that nearly all liquids would undergo a transition to aglassy state if crystallization is bypassed on cooling. A necessary andsufficient condition for this transition is that the liquid solutionshould be rapidly cooled to the glass transition temperature so thatnucleation and crystal growth cannot occur. Typically, the requisitecooling rates are very high for water (approximately 10⁷° C./min), butthey can be reduced to more workable levels (approximately 10° C./min)by the addition of cryoprotectants (CPA, usually 50 to 60% w/w).However, CPA concentrations this high are typically lethal to biologicalcells. New methods of ultra-rapid cooling are needed to achieve glassystate during cooling of biological cell suspensions.

The formation of intracellular ice during freezing may be avoided byhyperquenching the cells. In hyperquenching the water is cooled soquickly that nucleation events do not occur and the liquid undergoes aglass phase transition. As liquid water is cooled below its freezingpoint, it becomes energetically favorable for nucleation to occur. At130K, however, liquid water goes through a glass phase transition, whichis a second order thermodynamic phase transition, and the relaxationtime for the molecules becomes greater than laboratory time scales—i.e.,the viscosity of the fluid increases so that molecular rearrangementinto crystals becomes impossible. If one can get water to the glassphase before ice crystals nucleate, then one creates an amorphous solidreferred to as either amorphous solid water or amorphous ice.

There are a number of ways to form glass phase solid water; water vapordeposition on to cryo-cooled plates at very low pressures, exposingcrystalline ice to very high pressures at temperatures below 130K andthereby crushing it to the glass phase, and spraying watermicro-droplets at supersonic velocities onto cryoplates. None of thesetechniques, however, is suitable for use in freezing cells. Water vapordeposition simply cannot be done with a cell, and the other two methodsexpose the cell to lethal stress.

In order to preserve the wide variety of cellular material needed incurrent medical procedures, a new method of ultra rapid cooling that cansuccessfully cryopreserve biological material without the formation ofintracellular ice is needed. Such a method must avoid the use of highconcentrations of CPAs which are toxic to many cell types and not exposethe biological material to damaging physical stresses.

SUMMARY OF THE INVENTION

The technique of ultra-rapid freezing uses spatially confined heating tohold tissue samples warm while freezing the surroundings to cryogenictemperatures. Upon removal of the heating the sample freezes veryrapidly due to direct thermal contact with the surrounding material.Numerical calculations indicate that freezing rates of up to 1 milliondegrees per second can be achieved with this technique. This freezingrate is so fast that the sample will not freeze into a crystalline solidsuch as ordinary ice, but into a glass phase solid that has no crystals,i.e., the sample is vitrified.

The method includes placing a cellular material sample in thermalcontact with a cryogenically coolable environment. The cryogenicallycoolable environment is maintained at a temperature below the glassphase transition temperature of the cellular material while at least aportion of the cellular material is maintained at physiologicaltemperatures by spatially confined heating. The spatially confinedheating is then stopped resulting in the cooling of the cellularmaterial. This method results in cooling rates greater than 10⁵° C. persecond, and even cooling rates greater than 10⁶° C. per second,resulting in the vitrification of the cellular material.

In specific embodiments, the spatially confined heating may be providedusing a radiant energy source that is at least partially absorbed by thecellular material or by the water found within the cellular material.The cryogenically coolable environment may be provided by a cryostageor, in one embodiment, by suspending the cellular material within a holeformed in a high thermal conductivity material.

Material cryopreserved using the method of the invention may berecovered by warming the material at a warming rate sufficiently high soas to prevent devitrification, i.e. the nucleation of ice crystals fromthe glass phase during thawing.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more fully understood by reference to thefollowing detailed description when considered in conjunction with theaccompanying drawings, in which:

FIG. 1 illustrates a step in the method of the invention whereinspatially confined heating is provided by radiant energy supplied tosuspended biological tissue;

FIG. 2 illustrates a step in the method of the invention whereinspatially confined heating is provided by a focused beam of radiantenergy to a biological tissue sample located on a cryostage over anadiabatic region;

FIG. 3 illustrates a step in the method of the invention whereinspatially confined heating is provided by a focused beam of radiantenergy to a biological tissue sample located in a well formed in a highthermal conductivity material that has been cooled;

FIG. 4 illustrates diagrammatically the step shown in FIG. 2;

FIGS. 5A and B illustrate top and side views, respectively, of acryostage useful with the method of the invention;

FIG. 6 illustrates a sequence of steps for computing a solution for anumerical model of the method of the invention;

FIG. 7 graphs isotherms for a radiant energy warmed biological tissuesample on a cryostage against the radius (R) and height (Z) of thesample;

FIG. 8 graphs isotherms for a radiant energy warmed biological tissuesample on an adiabatic region on a cryostage against the radius (R) andheight (Z) of the sample;

FIG. 9 shows photographs of a 0.5 Molar sucrose solution after lasermelting and rapid solidification, the sample on the left has been frozento −150° C., the same sample is shown on the right having“recrystallized” at −30° C.;

FIG. 10 shows a sample slide constructed for use in holding a diluteaqueous solution for rapid freezing;

FIGS. 11A and B illustrate a sample holding apparatus for use inpreparing and testing rapidly frozen samples;

FIGS. 12A and B illustrate x-ray diffraction results for a crystallineice sample (A) and a rapidly frozen amorphous ice sample (B); and

FIGS. 13A and B plot maximum intensity of x-ray diffraction images as afunction of diffraction angle for a crystalline ice sample (A) and arapidly frozen amorphous ice sample (B).

DETAILED DESCRIPTION OF THE INVENTION

A cryopreservation method of the invention includes using radiant energyto provide spatially confined heating to a region of biological materialwhile cooling the surrounding environment to temperatures below theglass phase transition temperature of the biological material. The glassphase transition temperature is about 130K for pure water, higher foraqueous solutions. Accordingly, the glass phase transition temperaturefor the biological material will be about 130K, but may be higher ifintracellular cryoprotective agents (CPAs) are added. Sudden removal ofthe heat source results in cooling rates so fast that the intracellularsolution forms a non-crystalline glass phase without forming damagingice crystals. The cryogenically frozen biological material sample canthen be warmed at moderate to fast warming rates so that devitrification(the nucleation of crystals from the glass phase) does not occur duringthawing, and the entire cryopreservation process can be completedwithout forming ice crystals inside the cell.

Spatially confined warming of a biological tissue sample can be achievedin a number of ways. As illustrated in FIG. 1, the biological tissue 10can be suspended in a solution 12 that is placed into contact with acryogenic cooling plate 14. The spatially confined warming can beprovided by applying radiant energy 16 that is selectively absorbed bythe tissue 10 and not by the surroundings. For example, the radiantenergy 16 may include the use of laser energy having a wavelength of 577nm to target erythrocytes as a suspended biological tissue sample 10 forwarming in a saline solution 12—the erythrocytes absorbing energy havingthat wavelength while the surroundings do not. Alternatively, 2 micronradiation can be used to target the water in biological tissue 10surrounded by a non-absorptive material such as silicon oil 12.

In the described spatial heating configuration, the heating only occursin the biological tissue 10 and the tissue 10 can be held atphysiological temperatures (between approximately 0 to 40° C.) while thesurroundings are cooled by cryoplate 14. When the spatially confinedheating is removed, by stopping the application of the radiant energy 16to the biological tissue, the biological tissue 10 cools rapidly. Usingthe cryopreservation method of the invention, resulting cooling ratescan be as high as 1 million degrees per second for a 10 micron diameterbiological tissue sample. The cooling rate will decrease as the diameterof the sample squared increases.

Another way in which spatially confined heating can be achieved is touse a strongly focused beam of energy that is absorbed by both thebiological tissue and surrounding medium. One method for cryopreservingbiological material of the invention using this approach is illustratedin FIG. 2. Biological tissue sample 20 can be cooled from below by asurface 22 that has an adiabatic center region 24 while a focused beamof laser or microwave energy 26 maintains at least a portion of thesample at physiological temperatures.

In an additional method of the invention illustrated in FIG. 3,biological tissue sample 30 is cooled peripherally by being injectedinto a hole 32 that has been drilled into a material having a highthermal conductivity 34 that is cooled to cryogenic temperatures.Biological tissue sample 30 can also be maintained at physiologictemperatures by applying a focused beam of laser or microwave energy 36to the sample 30. Numerical experiments indicate that a cylindricalregion of the sample can be held between 0 and 37° C. if the sample iseither cooled from below by a surface that has an adiabatic centerregion, or if the sample is cooled peripherally by being injected into ahole that has been drilled in a high conductivity material. Once againcooling rates of up to 1 million degrees per second can be achieved forregions 10 microns in diameter when the radiant energy is suddenlyremoved.

By using planar heat sources and computer controlled stages, thistechnique could be used to freeze large numbers of cells or tissuesamples.

Once vitrified, samples may be stored at cryogenic temperatures. Asample may be recovered by warming at moderate to fast warming rates sothat devitrification (the nucleation of crystals from the glass phase)will not occur. Thus, the entire cryopreservation process can becompleted without any crystals forming inside the cell.

EXAMPLE 1 Numerical Model

Construction of a numerical model answers two fundamental questionsabout the cryopreservation method of the invention. The first is howlarge a sample region can be held between 0 and 40° C. by laser warmingwhile the surroundings are cooled to T_(c). The second is how fast thecooling rate will be once the laser heating is removed.

The basic geometry of the computational system is illustrateddiagrammatically in FIG. 4. Cells which are either in suspension orattached to a glass surface as a sample 40 are cooled from below on acryostage 42 at a temperature T_(c). An example of such a sample 40 andcryostage 42 is shown in FIGS. 5A and 5B. Cryostage 42 may suitably beconstructed of Lexan and is designed specifically to preventcondensation on the sample surface so that laser energy is not divertedfrom the sample 40. Nitrogen (N₂) gas is forced through a copper coilimmersed in liquid nitrogen (not shown). The cold N₂ gas then flowsthrough an insulated tube 44 to the cryostage 42 where it enters aninternal channel 46 and passes under the sample. The cold N₂ gas is thenredirected back to exhaust over the top of the sample 40 where itcreates a moisture free layer. In this way, sample 40 can be cooled to−196° C. without condensation forming on the sample surface.

Returning to the numerical model, the top 48 of sample 40 is exposed togas and thus will have negligible heat transfer compared to that throughthe conducting bottom surface. As sample 40 is cooled, a laser 50 holdsa small region of the sample at a temperature between 0 and 40° C. sothat the cells are neither damaged by excessive heat nor frozen.

The system is modeled as an axi-symmetric two-dimensional system incylindrical coordinates. The conservation of energy equation can benondimensionalized to the form: $\begin{matrix}{{\overset{\sim}{c}\frac{\partial\theta}{\partial\tau}} = {{A^{2}\frac{\partial}{\partial Z}\left( {\overset{\sim}{k}\frac{\partial\theta}{\partial Z}} \right)} + {\frac{\partial}{\partial R}\left( {\overset{\sim}{k}R\frac{\partial\theta}{\partial R}} \right)} + \overset{\sim}{Q}}} & (1)\end{matrix}$

where the dimensionless variables are: $\begin{matrix}{R = {{\frac{r}{R_{0}}\quad Z} = {{\frac{z}{H}\quad \tau} = {{\frac{\alpha\tau}{R_{0}^{2}}\quad A} = \frac{R_{0}}{H}}}}} & (2) \\{\theta = {{\frac{\left( {T - T_{c}} \right)}{\left( {T_{0} - T_{c}} \right)}\quad \overset{\sim}{k}} = {{\frac{k}{k_{f}}\quad \overset{\sim}{Q}} = \frac{{q\&}\quad R_{0}^{2}}{k_{f}\left( {T_{0} - T_{c}} \right)}}}} & (3) \\{\overset{\sim}{c} = \frac{\rho \quad c}{\rho \quad f_{f}}} & (4)\end{matrix}$

In these equations R₀ is the radius of the laser heating, k_(f), c_(f)and ρ_(f) are the thermal conductivity, specific heat and density of thefluid phase. T_(c) is the temperature of the cold boundary. T₀ is theequilibrium melting temperature of the ice. H is the height of theliquid/ice layer (the thickness of the layer) and q is the rate ofvolumetric heating due to the laser. The material properties are modeledas phase dependent, but not temperature dependent within a single phase.The thermal conductivity of crystalline ice is modeled as 4 k_(f) andthe product ρ_(c) of the crystalline ice was modeled as ρ_(f)c_(f)/2[White, F. M., Heat and Mass Transfer, Addison-Wesley, Reading, Mass.(1998)]. It is not clear whether the effect of modeling the thermalproperties as temperature dependent in addition to phase dependent wouldincrease or decrease the calculated freezing rate since both k and ctend to decrease with temperature. The thermal conductivity of liquidwater, moreover, changes by less than a factor of two over the 400Kelvin degrees for which data is available [Bejan, A., Heat Transfer,Wiley and Sons, New York, N.Y. (1993)]. Neglecting the temperaturedependence, therefore, will have only a small effect on the calculatedcooling rates.

The system is heated volumetrically by the laser in the region R<1.Uniform heating is assumed within the irradiated region. In the actualsystem, the heating would decrease with depth as the laser energy isabsorbed; but approximately uniform heating can be achieved by selectinga laser wavelength with a small coefficient of absorption. Thedisadvantage of this technique is that it requires increased laserpower. In the region R>1, there is no volumetric heating. The firstboundary conditions studied were negligible heat transfer at the topsurface (Z=1) and an isothermal bottom surface (Z=0) at temperatureT_(c) (θ=0). A symmetry condition is imposed at R=0 and T=T_(c) at theedge of the computational domain R=R_(edge).

In order to solve for the cooling rate once the laser has been turnedoff (Q=0), an initial condition must be available that is the steadystate solution with the laser turned on. This steady solution must besuch that there is a fluid region in which cells would not becomecrystallized (T>0° C.), but the maximum temperature must not be so hotthat the cells are damaged (less than 40° C.). Q must be adjusted untilthe maximum temperature in the system is approximately 40° C.

The sequence of techniques used in computation of the solution isillustrated in FIG. 6. The steady state solution is developed in twosteps. In the first step, a combined spectral and finite differencemethod is used to obtain a rough solution. If the thermal conductivityis assumed to be constant throughout the computational domain, thedimensionless temperature can be represented by a Fourier series of theform: $\begin{matrix}{\theta = {\sum\limits_{{n = 1},3,5}\quad {{a_{n}(r)}\quad \sin \quad \left( {\frac{n\quad \pi}{2}Z} \right)}}} & (5)\end{matrix}$

Plugging this into the steady state energy conservation equation andmaking use of the orthogonality of sines results in a second orderordinary differential equation (ODE): $\begin{matrix}{{{A^{2}\frac{{a_{n}\left( {n\quad \pi} \right)}^{2}}{4}} - \frac{{{}_{}^{}{}_{}^{}}}{R^{2}} - \frac{1{a_{n}}}{R{r}}} = {\frac{4}{n\quad \pi}\overset{\_}{Q}}} & (6)\end{matrix}$

The boundary conditions in R become: $\begin{matrix}{\frac{a_{n}}{r} = {{0\quad {at}\quad R} = 0}} & (7)\end{matrix}$

and

a_(n)=0 at R=R_(edge)  (8)

The ODE is solved by using finite difference discretization and taking15 modes in the series. The resulting linear system of equations issolved using a successive over-relaxation (SOR) iterative scheme.

This solution and the Q value found are then used as an initialcondition for a two dimensional finite-difference scheme in which thethermal conductivity is modeled as phase dependent. The two-dimensionalfinite-difference system with variable conductivity is solved using analternating direction implicit (ADI) method, a second order accuratetime-stepping scheme that can be used for both steady state andtransient analysis. The convergence criteria for the steady statesolution is that the maximum relative error be less than 0.001.

The cooling rate when the laser is turned off is calculated using thesame ADI finite difference scheme. The cooling rate is defined as theinstantaneous rate of cooling when the temperature of a liquid regiondropped below θ=0.27 (T=130K).

The reported solutions are generated using ΔR=0.2 and ΔZ=0.2, Δτ=0.001and R_(edge)=4+R_(adiabatic) (R_(adiabatic) is an adiabatic centerregion that is necessary for the computation and is described below).Convergence tests indicate that the calculated cooling rates change byless than 0.5% when ΔR and ΔZ are halved, and by less than 2% when Δτ isreduced by a factor of 10. R_(edge) is chosen so that less than 2% ofthe total heat transfer occurred through the R=R_(edge) surface.

The combined finite difference and spectral method converges veryquickly. The convergence criteria is set as the maximum error beingsmaller than 1e−4. Different Q values are tried until a value of Q isfound that gives a maximum temperature of about θ=1.2, which correspondsto 39.2° C. if T_(c)=77K and T₀=273K. The Q value and steady statesolution are then refined using the ADI solver with phase dependentconductivity. Q is adjusted until θ_(max) was within 3% of 1.2.

Isotherms of the steady state solution found using this technique foraspect ratio A=1 are shown in FIG. 7. The region inside the firstisotherm (θ>1) is liquid water. It can be seen from FIG. 7 that having abottom boundary held at 77K results in a region of liquid water that istoo small in which to reliably place a cell. Changing the aspect ratiodoes not result in a more suitable liquid region.

In order to increase the size of the liquid region, the bottom boundarycondition is changed so that an adiabatic region of radius R_(adiabatic)extends out from the center of the sample. FIG. 8 illustrates theisotherms when the aspect ratio A=1 and the adiabatic region radius istwice that of the laser beam, i.e., R_(adiabatic)=2. One can see thatthe liquid region extends all the way to the bottom of the sample. Thisis a suitable liquid region in which to place cells reliably.

This steady state solution is used as the initial condition for the ADItime-stepping scheme used to calculate cooling rates. The instantaneouscooling rates are calculated for points originally in the liquid regionas they pass through the glass phase transition temperature θ_(g)=0.27.The dimensionless cooling rates are fairly uniform throughout the liquiddomain at 1.68. If the laser beam diameter is taken to be 10 microns,this indicates a cooling rate of greater than 10⁶K per second.

One method for creating a sample with an adiabatic center region is todrill a hole in the sample slide with hydrophobic walls so the sampleliquid would not creep down into the hole. Alternatively, a hole couldbe drilled into a high conductivity material such as platinum ofsapphire (which are both non-toxic to the cells) and the sample could beinjected into the hole. Numerical simulation of this configuration wasperformed as well and it was found that these two techniques result inalmost identical cooling rates.

The numerical models indicate, therefore, that by careful choice ofgeometry a cell can be held warm by a laser while the surroundings arecooled to 77K.

EXAMPLE 2 Freezing of Dilute Aqueous Solutions

Experiments show that cooling rates similar to those predicted with thenumerical model can be achieved during the freezing of dilute aqueoussolutions. These experiments demonstrate that one can achieve anamorphous solid ice using laser warming with solutions that are muchmore dilute than those currently required to achieve vitrification withconventional freezing techniques.

The samples consist of a layer of aqueous solution sandwiched betweentwo parallel glass plates separated by distances from 10 to 100 microns.The solutions used in these experiments are sucrose solutions rangingfrom 0 to 2 molar. In addition to the sucrose, 0.015 M of Amaranth (FD&CRed Dye #2) are added. The dye is necessary because the laser usedproduces a beam at 532 nm which is not absorbed by water.Spectrophotometric analysis indicates that 0.015 M Amaranth would cause10% to 50% of the beam energy to be absorbed in passing through theliquid layer, depending on the layer thickness.

The sample was placed on cryostage 42 (FIGS. 5A and 5B) constructed ofLexan. Temperature of the sample can be monitored by a type Tthermocouple imbedded in the ice. When the temperature reaches thedesired level, the laser is fired. The laser is capable of delivering a400 mJ pulse in 7 ns. The energy delivered per pulse is adjusted untilthe firing of the laser results in a clear region without gas bubbles.The sample is then warmed by reducing the gas flow rate, or by reducingthe contact area between the copper coil and the liquid nitrogen.

Initial experiments demonstrated that a clear region could be createdfor solutions of 2M, 1M and 0.5M sucrose. FIG. 9 provides pictures takenof clear regions produced by laser warming and rapid pictures taken ofclear regions produced by laser warming and rapid refreezing of 0.5Msucrose solution. The clear regions are completely transparent, as isliquid water, but at −30° C. the ice becomes opaque once again. Thereturn to opacity at temperatures well below the melting point indicatesthat the clear regions are not a result of photo-bleaching or some laserinduced chemical reaction. The ice appears to return to its originalstate.

EXAMPLE 3 Rapid Freezing of Dilute Aqueous Solutions with X-rayDiffraction Analysis

A testing method and apparatus have been devised to provide x-raydiffraction analysis of solutions cooled by the method of the invention.In the test procedure, the sample is cooled to below the glass phasetransition temperature of the solution (130K for pure water, higher foraqueous solutions) by submerging it in liquid nitrogen and maintained atthat temperature. A portion of the sample is laser warmed. The laser isthen turned off, and the warmed portion of the sample is rapidly cooledas described above. The sample must then be transferred into the x-raydiffractometer without raising the temperature above 150K. Often,transfer of the samples to the diffractometer results in exposure of thesample to room air which causes crystalline ice to immediately form onthe sample. To avoid this a sample holder is used that can be placeddirectly into the diffractometer without exposing the sample to roomair. X-ray diffraction results may then confirm the presence ofamorphous ice produced by the method.

Rapid solidification of the dilute aqueous solutions is accomplished byfirst injecting solution into a channel 60 of known height H between twoglass coverslips 62, 64 to create a sample slide 66 as shown in FIG. 10.The height of channel 60 is controlled by mixing cross-linkedpolystyrene microbeads 68 of known diameter into a UV curing glue. Theglue is spread along the edges of one coverslip 62 and the secondcoverslip 64 is pressed onto the first. The glue is then cured byexposure to a UV lamp. The height of the channel 60 can be measured byusing a micrometer to measure the total height of the resulting slideand subtracting the thickness of the individual coverslips 62, 64.

The aqueous solutions are mixtures of distilled water and sucrose. Inorder to selectively heat the aqueous solution, a laser pulse 70 at 532nm is used. The aqueous solutions also contained 0.005 M ameranth (FD&CRed Dye #2) which absorbs strongly at 532 nm. By testing varying dyeconcentrations over a large range it was determined that the absorbanceof 532 nm radiation was linearly related to dye concentration. Based onthis relationship, a 0.005 M dye solution absorbs less than 5 % of theenergy incident on a 10 micron layer. This low absorbance was selectedso that roughly uniform heating could be achieved throughout the layerof solution in the sample slide 66. The laser used to deliver the pulseis a Q-switched YAG capable of delivering 400 mJ of energy in a 7nanosecond pulse.

The sample holding apparatus 70 used consists of a Dewar flask 72 with acopper J-bar 74 that could be lowered (FIG. 11A) and raised (FIG. 11B)in and out of a liquid nitrogen bath 76. A copper clamp machined intothe end of the J-bar 74 holds the sample 66. The apparatus 70 includes ahole 78 through which the laser and the x-ray beam can be aimed. Whenthe sample 66 is in a raised position, a supplementary flow of coldnitrogen gas 80, provided by either the x-ray diffractometer or by anauxiliary tank, creates a strong outward flow of nitrogen gas throughhole 78 that prevents condensation from accumulating on the sample 66.Experiments performed with thermocouples imbedded in glass slides showthat the sample temperature remains within 5° C. of the gas temperaturefor temperatures cooler than −100° C.

Sample 66 is held in the raised position to deliver the laser energythrough hole 78. Nitrogen gas 80 is forced through a copper coilimmersed in liquid nitrogen and injected into the apparatus. The gastemperature near the sample was measured via thermocouple and the gasflow rate adjusted until the gas temperature was −165° C. The lasersupplied a beam of energy focused to a diameter of 2.8 mm and Q-switchedpulses were delivered at different power settings until a region ofclear ice formed in the sample 66 that did not contain bubbles. A laserfluence of about 1.1 J/cm² was optimal. After a clear region is createdin the sample 66, the sample is lowered into the liquid nitrogen bath 76and the sample holding apparatus 70 is transported to the x-raydiffractometer.

A Seimens SMART System CCD diffractometer was used for the x-rayanalysis. The built-in nitrogen gas supply temperature was adjusted to−165° C. and the sample holding apparatus 70 was inserted into thediffractometer. The built in nitrogen gas supply was connected to thetop of the apparatus 70 and the sample 66 was raised into position andadjusted so that the x-ray beam struck the sample in a control region ofcrystalline ice so that a baseline crystal pattern could be recorded.The sample 66 was then repositioned so the beam passed through therapidly solidified ice. X-ray diffraction patterns were recorded on a6.18 cm CCD using a 60 second exposure. The x-ray beam was a 0.5 mmcollimated beam from a Molybdenum source.

FIGS. 12A and 12B show the x-ray diffraction images obtained from aregion of pure crystalline ice (12A) and laser created amorphous ice(12B) at different positions of the same slide having a sample of 1 Msucrose solution and a channel height H (FIG. 10) of 21 microns. Thebroad amorphous scattering in both images is a result of the glassslides surrounding the ice, but bright spots appearing in FIG. 12A bothwithin the ring of diffuse scattering and beyond it indicate thepresence of crystals. The lack of apparent bright spots in FIG. 12Bsuggests that the sample is amorphous. In order to determine whether anycrystalline peaks were present in the laser treated region, the x-rayimage was analyzed by a computer code that searched for the maximumintensity within the sample at each radial position. Plots of maximumintensity (arbitrary units) as a function of diffraction angle are shownin FIGS. 13A and 13B.

The characteristic peaks exhibited by crystalline ice are clearlyvisible in the crystalline sample (FIG. 13A), but these peaks do notappear in the laser treated sample (FIG. 13B). The laser melted andrapidly solidified ice does not show evidence of any crystalline peaksat the locations corresponding to the peaks of crystalline ice. Thesmall peaks at various other angular locations may indicate smallamounts of condensation on the surface of the slide, but the completelack of spikes corresponding to the spikes in the crystalline ice sampleproves that the sample is completely amorphous.

Additional samples studied show that the method produces amorphous icein less dilute aqueous solutions, and even in pure water.

EXAMPLE 4 Vitrification of Human Erythrocytes

Applying a process similar to those used in Examples 2 and 3, a solutioncontaining human erythrocytes was first frozen, then warmed with radiantenergy and rapidly refrozen to vitrify the erythrocytes. Unprotectedhuman erythrocytes were diluted in a phosphate buffered saline andbovine calf serum solution and frozen at a cooling rate of about 10,000°C./minute, resulting in a large amount of intracellular ice formation. Agroup of frozen cells were treated with conventional thawing procedureswith warming rates of up to 80,000° C./minute—all of these cells lysed.

Another group of frozen cells were treated with a pulse from aQ-switched YAG laser at 532 nm so that the intracellular solution (whichabsorbs 532 nm radiation) melted; but the extracellular solution (whichis transparent to 532 nm radiation) did not melt. The vitrificationstudies described in Examples 2 and 3 above show that this lasertreatment leads to vitrification. The laser treated cells were thenthawed using conventional protocols and 80 % of the cells remainedintact.

It will be understood that the foregoing is only illustrative of theprinciples of the invention, and that various modifications can be madeby those skilled in the art without departing from the scope and spiritof the invention. All references cited herein are expressly incorporatedby reference in their entirety.

What is claimed is:
 1. A method for vitrifying biological materialcomprising: (a) placing a biological material having a glass phasetransition temperature in thermal contact with a cryogenically coolableenvironment; (b) cooling the cryogenically coolable environment to atemperature below the glass phase transition temperature of thebiological material; (c) simultaneously with the cooling of theenvironment in thermal contact with the biological material, applyingspatially confined heating using radiant energy to the biologicalmaterial to maintain the temperature of at least a portion of thebiological material at a physiological temperature of between about 0 to40°; (d) stopping the application of spatially confined heating to thebiological material to rapidly cool the and thereby vitrify thebiological material.
 2. The method of claim 1, wherein the cryogenicallycoolable environment is cooled to a temperature less than or equal toabout 130 Kelvin degrees.
 3. The method of claim 1, wherein stopping theapplication of spatially confined heating results in a cooling rateequal to or greater than about 10⁵° C./second for the biologicalmaterial.
 4. The method of claim 1, wherein stopping the application ofspatially confined heating results in a cooling rate equal to or greaterthan about 10⁶° C./second for the biological material.
 5. The method ofclaim 1, further comprising the step of recovering the vitrifiedbiological material by warming the material at a sufficiently high rateto prevent the occurrence of devitrification.
 6. The method of claim 1,wherein the radiant energy is at least partially absorbed by waterwithin the biological material.
 7. The method of claim 6, wherein thebiological material is suspended in a medium that does not absorb theradiant energy.
 8. The method of claim 1, wherein the radiant energy isat least partially absorbed by the biological material.
 9. The method ofclaim 8, wherein the biological material is suspended in an aqueoussolution.
 10. The method of claim 1, wherein the radiant energy is inthe form of a focused beam aimed toward at least a portion of thebiological material.
 11. The method of claim 10, wherein the radiantenergy is supplied using a laser.
 12. The method of claim 10, whereinthe biological material is suspended in thermal contact with acryostage.
 13. The method of claim 12, wherein there is an adiabaticregion between at least a portion of the biological material suspensionand the cryostage.
 14. A method for vitrifying cellular materialcomprising: (a) suspending a cellular material having a glass phasetransition temperature in a solution to form a cellular materialsolution; (b) cooling the cellular material solution to a temperaturebelow the glass phase transition temperature of the cellular materialwhile maintaining at least a portion of the cellular material at atemperature between about 0C and 40° C.; and (c) rapidly cooling theportion of the cellular material maintained at between about 0° C. and40° C. to a temperature at or below the glass phase transitiontemperature of the cellular material at a cooling rate greater thanabout 10⁵° C./second so as to vitrify the cellular material.
 15. Themethod of claim 14, wherein the cooling rate is greater than about 10⁶°C./second.
 16. The method of claim 14, wherein the cellular materialsolution is cooled by placing the cellular material solution in thermalcontact with a cryogenically coolable environment that is maintained ata temperature below the glass phase transition temperature of thecellular material.
 17. The method of claim 16, wherein the cellularmaterial is maintained at a temperature between about 0° C. and 40° C.by applying spatially confined heating to the cellular materialsolution.
 18. The method of claim 17, wherein the cellular material israpidly cooled by suddenly stopping the spatially confined heating. 19.The method of claim 17, wherein the spatially confined heating consistsof radiant energy directed to the cellular material solution.
 20. Themethod of claim 19, wherein the radiant energy is in the form of a beamfocused on at least a portion of the cellular material solution.
 21. Themethod of claim 19, wherein the radiant energy is of a wavelength thatis at least partially absorbed by the cellular material.
 22. The methodof claim 19, wherein the radiant energy is of a wavelength that is atleast partially absorbed by intracellular water within the cellularmaterial.
 23. The method of claim 14, wherein at least a portion of thecellular material is cooled to below about 130 Kelvin degrees.
 24. Themethod of claim 14, wherein the cellular material solution is in thermalcontact with a cryostage.
 25. The method of claim 24, wherein there isan adiabatic region between at least a portion of the cellular materialsolution and the cryostage.
 26. The method of claim 14, wherein thecellular material solution is in thermal contact with a cryogenicallycoolable environment by being suspended within a hole formed in a highthermal conductivity material.